Loss of SEC-23 in Caenorhabditis elegans causes defects in oogenesis, morphogenesis, and extracellular matrix secretion

Abstract

SEC-23 is a component of coat protein complex II (COPII)-coated vesicles involved in the endoplasmic reticulum-to-Golgi transport pathway of eukaryotes. During postembryonic life, Caenorhabditis elegans is surrounded by a collagenous exoskeleton termed the cuticle. From a screen for mutants defective in cuticle secretion, we identified and characterized a sec-23 mutant of C. elegans. By sequence homology, C. elegans has only the single sec-23 gene described herein. In addition to the cuticle secretion defect, mutants fail to complete embryonic morphogenesis. However, they progress through the earlier stages of embryogenesis, including gastrulation, and achieve substantial morphogenesis before death. We demonstrated a maternal component of SEC-23 function sufficient for progression through the earlier stages of embryogenesis and explaining the limited phenotype of the zygotic mutant. By RNA-mediated interference, we investigated the effects of perturbing COPII function during various postembryonic stages. During larval stages, major defects in cuticle synthesis and molting were observed. In the adult hermaphrodite, reduction of SEC-23 function by RNA-mediated interference caused a rapid onset of sterility, with defects in oogenesis including early maturation of the germline nuclei, probably a result of the observed loss of the GLP-1 receptor from the membrane surfaces adjacent to the developing germline nuclei.

Figure 1.

ij13 genomic location, sec-23 gene structure, and sec-23 mini-gene. (A) Extreme right-hand end of chromosome V showing the position of the ij13 mutation. (B) Genomic organization of sec-23 (predicted gene Y113G7A.3). The black boxes represent exons. The position of the base change in ij13 is indicated. (C) Schematic representation of the sec-23 mini-gene (see MATERIALS AND METHODS for details).

Figure 2.

Sequence comparison of SEC23 proteins. Predicted protein sequences were aligned using the AlignX program of VectorNTI. The sequences were obtained from databases at the European Bioinformatics Institute (http://www.ebi.ac.uk/), with the following accession numbers: C. elegans, Q9U2Z1; D. melanogaster, Q9VNF8; human (SEC23A), Q15436; and S. cerevisiae, P15303. Residues shared among all four sequences are shown as white on black; residues shared between two or three of the sequences are shown as black on gray. The residue affected by the ij13 mutation is marked with ^*.

Figure 3.

Morphogenesis in sec-23(ij13) embryos. (A–D) Elongation of a wild-type embryo: comma stage (A), two-fold stage (B), three fold stage (C), and pretzel stage (D) after secretion of the cuticle. (E–I) Elongation and collapse of a sec-23(ij13) embryo. (E–H) Images taken at 30-min intervals. (I) Image taken 90 min after H. White arrows, anterior and posterior ends of the pharynx (anterior to the left); black arrows, the width of the pharynx at various points. (J–L) Images taken at 150-min intervals of a homozygotic sec-23(ij13) mutant embryo derived from a chromosomally sec-23(ij13) homozygous mutant mother, transgenic for sec-23. In J, the animal has just initiated hypodermal morphogenesis.

Figure 4.

Effect of sec-23 mutation on pharyngeal development and on the secretion of collagen and a basement membrane marker. (A and B) A pretzel-stage wild-type embryo (A) and a sec-23(ij13) mutant (B) were stained with antibody 3NB12, which labels pharyngeal muscle cells (Priess and Thomson, 1987). The anterior (a) and posterior (p) pharyngeal bulbs are indicated in A; the two bulbs are also indicated in B, but due to the severity of the morphological defects, it is not possible to distinguish anterior and posterior. (C–F) Immunolocalization of the DPY-7 collagen with the DPY7–5a mAb. (C) A wild-type late comma-stage embryo showing perinuclear localization of DPY-7 in hypodermal cells before secretion. Two nuclei are indicated by white arrows. (D) A wild-type pretzel-stage embryo with DPY-7 in the cuticle. (E) A sec-23(ij13) mutant embryo at terminal zygotic phenotype, with some DPY-7 retained in a perinuclear location (arrow) and most in aberrant strands or clumps. (F) Perinuclear DPY-7 (arrows) in a sec-23(ij13) mutant embryo derived from a chromosomally sec-23(ij13) homozygous mutant mother transgenic for sec-23. (G) Nonperinuclear localization of the basement membrane component perlecan with MH3 antiserum in a mutant embryo like that in F. (H and I) Localization of DPY-7 to circumferential bands in the cuticle of a wild-type larva (H) and to discontinuous bands in a sec-23 RNAi larva.

Figure 5.

sec-23::GFP reporter gene expression and restoration of sec-23 function in the hypodermis. Nomarski images (left column) are of the same embryos shown in the fluorescence images. (A–D) Expression of sec-23::GFP (A and B) and dpy-7::GFP (C and D) reporter genes in the hypodermal cells of wild-type embryos at the one-and-a-half-fold (A and B) or comma (C and D) stage. (E–G) Rescue of morphogenesis and of late-stage cuticle collagen col-12::GFP reporter gene expression by a dpy-7::sec-23 gene fusion. (E and F) A homozygous sec-23(ij13) mutant embryo containing only the col-12::GFP reporter gene. (G and H) A mutant embryo containing also the dpy-7::sec-23 gene fusion.

Figure 6.

Subcellular localization of a SEC-23::GFP fusion protein. All images are of the same embryo. (A and B) Images in the same focal plane showing SEC-23::GFP with anti-GFP antibody (A) and the ER resident enzyme PDI-2 (B). PDI-2 is detected as a halo surrounding the nuclei, which look like non-stained almost spherical structures. The arrows indicate the position of four hypodermal cells. (C–F) Higher magnification, composite images of the cells indicated in A and B, taken in four different focal planes. (C) Surface of the ER closest to the apical surface of the cells. (D–F) Sequentially deeper focal planes. The nuclei are clearly visualized in D and E but are almost out of vision in the focal plane of F (close to the basal side of the cells).

Figure 7.

Effects of sec-23 RNAi on larvae. (A) An untreated wild-type L4 larva. (B–F) Larvae subjected to sec-23 RNAi by the bacterial feeding method. (B) An animal of age similar to that in A, showing a lack of growth. (C) An animal that ruptured upon handling, demonstrating a weak cuticle. (D) An accumulation of undigested bacteria (arrows) in the intestine. (E) A molting defect; the head of an animal is stuck inside an old cuticle. (F) Bloated seam cells. Bars, 0.1 mm.

Figure 8.

Effect of sec-23 RNAi on the germ-line. Young adult hermaphrodites grown under normal culture conditions were transferred onto sec-23 RNAi bacterial lawns and allowed to feed for 18–48 h. The distal region (dist), turn (t), oocytes (ooc), spermatheca (sp), and in utero embryo (emb) are indicated. (A and B) Nomarski (A) and fluorescence (B) images of an adult hermaphrodite gonad expressing YP170::GFP. (C and D) Like A and B, but of an RNAi treated animal. (E and F) Fluorescence images of untreated (E) and RNAi treated (F) animals showing immunolocalization of the yolk receptor RME-2. (G and H) Nomarski images in two focal planes of an animal treated with RNAi for a few hours longer than those in C–F. (I–K) Fluorescence images of distal gonad arms stained with anti-GLP-1 antibody. Gonads are from an untreated animal (I) or from animals treated during adulthood for 18 h (J) or 36 h (K) with sec-23 bacterial-feeding RNAi. (L and M) Nomarski (L) and fluorescence (M) images of an animal after an RNAi treatment like that of G and H. A histone::GFP protein fusion expressed in the germline from a pie-1::GFP::H2B gene fusion permits visualization of chromatin. Arrows, two binucleate oocytes.